Fatty acid compositions

ABSTRACT

A natural or synthetic glycerol oil which comprises at least 20% by weight, preferably at least 25% of di-linoleoyl-mono-gamma-linolenyl-glycerol (DLMG), is used to supplement foods, nutritional compositions and in skin and hair care compositions.

Gamma-linolenic acid (GLA, 18:3 n-6) is of particular interest innutrition, in skin care and in the treatment of various diseases. GLA isa normal intermediate in human metabolism. It is derived bydelta-6-desaturation from the essential fatty acid (EFA) linoleic acid,which is found in substantial amounts in most diets. The conversionpath, showing common enzymes with the n-3 series of EFAs, is:

                  TABLE 1                                                         ______________________________________                                        n-6                   n-3                                                     ______________________________________                                        18:2 delta-9,12           18:3 delta-9,12,15                                  (linoleic acid)           (alpha-linolenic acid)                                           ##STR1##                                                         18:3 delta-6,9,12         18:4 delta-6,9,12,15                                (gamma-linolenic          (stearidonic acid)                                  acid)                                                                                      ##STR2##                                                         20:3 delta-8,11,14        20:4 delta-8,11,14,17                               (dihomo-gamma-                                                                linolenic acid)                                                                            ##STR3##                                                         20:4 delta-5,8,11,14      20:5 delta-5,8,11,                                  (arachidonic acid)        14,17                                                                         (`eicosapentaenoic                                                            acid`)                                                           ##STR4##                                                         22:4 delta-7,10,13,16     22:5 delta-7,10,13,16,19                            (adrenic acid)                                                                             ##STR5##                                                         22:5 delta-4,7,10,        22:6 delta-4,7,10,13,                               13,16                     16,19                                                                         (`docosahexaenoic                                                             acid`)                                              ______________________________________                                    

The above pathways are not normally reversible nor, in man, are n-3 andn-6 series acids inter-convertible.

The acids, which in nature are of the all-cis configuration, aresystematically named as derivatives of the corresponding octadecanoic,eicosanoic or docosanoic acids, eg. delta-9, 12-octadecadienoic acid ordelta-4, 7, 10, 13, 16, 19 docosahexaenoic acid, but numericaldesignations such as, correspondingly, 18:2 n-6 or 22:6 n-3 areconvenient. Initials, for example, EPA for the 20:5 n-3 acid(eicosapentaenoic acid) or DHA for the 22:6 n-3 acid (docosahexaenoicacid), are also used but do not serve when n-3 and n-6 acids of the samechain length and degree of unsaturation exist as for example with the22:5 acids. Trivial names in more or less common use in the n-6 seriesare as shown. Of the n-3 series only 18:3 n-3 has a commonly usedtrivial name, alpha-linolenic acid, though the name stearidonic acid iscoming into use for the 18:4 n-3 acid and the names eicosapentaenoicacid and docosahexanenoic acid as such are also used. The alpha isomerof linolenic acid was characterised earlier than gamma-linolenic acidand reference in the literature simply to linolenic acid, especially inthe earlier literature, is to the alpha-acid.

As is clear from the above, linoleic acid (LA) is an essential nutrientwhich, like a vitamin, cannot be made within the body and so must betaken in the diet. However, in order to be fully useful to the body, LAmust be metabolised, first to GLA and then to further metabolites.

LA has some functions which it can perform itself, particularly in theskin and other membranes and in relation to cholesterol transport, butmany of its effects require conversion to GLA and beyond. The GLA andthe further metabolites each appear to have particular roles in the bodyand so this first metabolic step is of particular significance. Itsimportance is emphasised by the fact that it seems vulnerable toblockade in a number of situations. For example, ageing, highcholesterol levels, high alcohol intake, certain viral infections,atopic eczema, breast pain, diabetic neuropathy and certain forms ofcancer all appear to be associated with a reduced ability to make GLA.In these situations, and in any other situation associated with a rateof GLA formation inadequate to supply the body's needs, it may beadvantageous to provide GLA directly.

Useful sources of GLA are relatively few. GLA is found in moderateamounts in human breast milk, but that is clearly not a practicalcommercial source. GLA can be synthesised with some difficulty and it ispossible that the known synthetic routes could be improved, or entirelynew ones found. A number of plant oils contain GLA, the most importantknown examples to date being the seed oils from the evening primrose(Oenothera spp), from borage (Borago officinalis) and from members ofthe Ribes family such as blackcurrants. Some fungi and algae formGLA-rich lipid stores, examples being strains of Rhizopus, Mortierella,Mucor and Spirulina.

The seed oil of the evening primrose (EPO) has been widely usedcommercially as a component of foods, as a nutritional supplement, as aningredient of cosmetic, skin care and hair care products, and as apharmaceutical. For example, it is used in Japan for addition toartificial infant milks to make them more like breast milk, while in theUnited Kingdom and some other countries it is approved as a prescriptiondrug for the treatment of atopic eczema and of breast pain. In manycountries throughout the world it is used as a nutritional supplementand as a skin product ingredient.

However, EPO contains only 8 to 10% of its fatty acids as GLA. It hastherefore been suggested that other sources such as borage oil, whichmay contain over 20% of GLA, or blackcurrant or fungal oils which maycontain in the region of 15 to 25% of GLA may provide better sources.However, it has somewhat surprisingly been found that clinically theseoils appear to be less effective than evening primrose oil, or not aseffective as would be expected given their GLA content. Many patientswho have experienced a clinical response to EPO, for example in the caseof atopic eczema or pre-menstrual breast pain, have relapsed onswitching to one of the other GLA sources, even though the amount of GLAconsumed may be greater. Biological testing has subsequently shown thatequal amounts of GLA may indeed have very different effects when thatGLA is supplied as different oil sources.

One of the most important biological consequences of administering GLAis its conversion within the body, first to dihomo-gamma-linolenic acid(DGLA) and then to prostaglandin (PGE1). PGE1 has a wide range ofdesirable effects, including being anti-inflammatory, vasodilator,anti-thrombotic, cholesterol-lowering and anti-hypertensive, and itsrelation to the n-6 series EFAs is shown in the following chart:##STR6##

In order to test their ability to stimulate the formation of PGE1, EPO,borage oil, blackcurrant oil and microbial oil were fed to laboratoryrats in amounts which provided the same daily dose of GLA. After thesame period on the diet, the mesenteric vascular bed was removed fromthe animals and its rate of production of PGE1 while being perfused withoxygenated buffer was measured. The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Amounts of PGE1 in ng/hour produced by the                                    perfused superior mesenteric vascular bed of rats fed                         with equal amounts of GLA per day in the form of evening                      primrose, fungal, blackcurrant or borage oils.                                Oil            PGE1 outflow                                                   ______________________________________                                        Evening primrose                                                                             14                                                             Fungal         6                                                              Blackcurrant   2                                                              Borage         1                                                              ______________________________________                                    

As can be seen, EPO was much more effective than the other oils atincreasing PGE1 outflow and since this effect of EPO is obviously notsimply related to the amount of GLA in the oil, it seemed to us that itmight depend on the detailed tri-glyceride structure of the oil. Fattyacids in vegetable oils are present not as the free fatty acidsthemselves but primarily as components of triglycerides, a glycerolbackbone ##STR7## with three fatty acids attached to it by ester links.It seemed to us possible that there might be a particular triglyceride(TG) present in EPO which happened to be there in much larger quantitiesthan in the other oils, and that this TG might account for the desirablebiological effects of EPO. We therefore quantitated the amounts of thevarious triglycerides present in each of the common sources of GLA, withresults set out in Table 3 appended to this specification.

The TGs in the oils were separated by reverse phase high pressure liquidchromatography (HPLC). We initially synthesised four triglycerides,tri-gamma-linolenyl-glycerol (tri-GLA), tri-linoleoyl-glycerol (tri-LA),mono-linoleoyl-di-gamma-linolenyl-glycerol (mono-LA-di-GLA) anddi-linoleoyl-mono-gamma-linolenyl-glycerol (DLMG). These were used tooptimise the conditions for analysis. The TGs were dissolved inchloroform and then applied to a Beckman System Gold (trade mark)programmable solvent module 126, with two identical 250 mm reverse phasecolumns (Supelcosil LC-18 (trade mark)) connected in series to increasethe resolution of the highly unsaturated TG species. The eluted TGfractions were detected by a mass (light scattering) detector suppliedby Applied Chromatography Systems, Macclesfield, England. Each fractionwas collected manually. Each fraction was then mixed with a known amountof internal standard (tri-heptadecanoin) and the methyl esters of thefatty acids were generated using BF₃ -methanol. The fatty acid methylesters were then analysed by gas chromatography using a 50 m fusedsilica capillary column (Supelcomega (trade mark)) in an HP 5890 gaschromatograph equipped with a flame ionisation detector. This techniqueclearly separates the triglyceride fractions and then identifies thefatty acids present in each fraction but it does not identify thepositions of the fatty acids in the TG molecule.

Using this technique, thirty five peaks were identified in borage oil,with the early peaks which contained the highly unsaturated fatty acidsbeing very clearly separated. There were only eighteen readilyidentifiable peaks in EPO. Blackcurrant and fungal oils which containmore fatty acids than EPO or borage oil contained many more peaks. Table3 shows all the identified peaks in EPO and borage oil.

Inspection of the table shows one particular GLA-containing fractionwhich is present at a high level in EPO but at a lower level in borageoil, the amount in EPO being substantially greater than the amountpresent in borage, with even lower levels in fungal and blackcurrantoils. This fraction contains two moieties of linoleic acid and one ofGLA (di-linoleoyl-mono-gamma-linolenyl-glycerol, (DLMG)). No otherGLA-containing peak shows such an obvious difference between the fouroils. DLMG makes up 18 to 19% of the triglycerides in EPO, smalleramounts of those in borage oil, and much smaller amounts in fungal orblackcurrant oils.

We have synthesized DLMG with its GLA in the 2 position or in the 1 or 3positions, and have found these molecules to be similar in their effectson elevated blood pressure in rats with spontaneous hypertension and oncholesterol levels in animals fed cholesterol. While there may be smalldifferences in biological effects depending on the precise position ofone GLA, it appears that the main beneficial effect relates to thepresence of two linoleic acid moieties and one GLA moiety on the sametriglyceride. This is thought to be because such a triglyceride willprovide LA which is positioned before the rate-limiting delta-6desaturase step in EFA metabolism, and GLA which comes after this step.Therefore, LGG, present at low levels in both EPO and borage oils isalso expected to have desirable biological effects. It is also expectedthat some effect may also be seen using TG's having one LA, one GLA anda different, third fatty acid residue, such as OLG and PLG, but theseare likely to be less valuable because of the presence of a moiety whichhas no essential fatty acid activity.

We therefore believe that it is the presence of DLMG which explains theusual biological potency of the GLA in EPO. This TG may have its GLA ineither the 2 position or in the 1 or 3 positions in the triglyceridemolecule, with linoleic acid occupying the other two positions.

Accordingly, the invention provides a triglyceride which isdi-linoleoyl-mono-gamma-linolenyl glycerol (DLMG).

On this basis, and herein lies one aspect of the invention, anypharmacologically or nutritionally acceptable natural or syntheticglyceride oil selectively enriched in or supplemented with DLMG is, inview of the established value of GLA and the now-shown value of itspresence as DLMG, a valuable and normal product both as such and in usein the preparation of a medicament or dietary composition for thepurposes for which GLA is valuable.

GLA itself has extensive potential uses as a pharmaceutical and as anutritional supplement. Many of these uses have been documented inprevious patents and patent applications from the present applicants.These applications relate to the use of GLA and other EFA's in thetreatment of a variety of disorders, including skin disorders such asatopic eczema, breast disorders including breast pain, premenstrualsyndrome, psychiatric disorders including alcoholism, schizophrenia andAlzheimer's disease, cardiovascular disorders including the preventionand treatment of coronary heart disease, peripheral vascular disease andhypertension, disorders of inflammation including rheumatoid arthritis,osteoarthritis, inflammatory bowel disease, connective tissue disordersand related conditions, disorders of immunity including multiplesclerosis, Sjogren's syndrome, systemic sclerosis and relatedconditions, diabetes and in particular the long term complications ofneuropathy, retinopathy, vascular disease and nephropathy, other renaldiseases, disorders of calcium and bone metabolism includingosteoporosis and excessive urinary calcium excretion, viral infectionsand post-viral fatigue syndrome, cancers, and the complicationsfollowing radiotherapy for cancer and other disorders. Nutritionalproblems of particular importance include enteral and parenteralsupplementation for infants, specialist infant formulae, supplements forthe elderly, and supplements of various types for those withgastrointestinal problems and malabsorption. This list of potentialapplications for the invention is not exhaustive and is provided as anexample of the possible utility of the invention.

Further, as far as we are aware, no evening primrose or other oil whichhas been used to date for nutritional, skin care, pharmaceutical orother purposes has contained more than 20% of DLMG. A further aspect ofthe invention is therefore an oil containing more than 20% by weight ofDLMG, desirably more than 25%, preferably more than 40%, very preferablymore than 60%, more preferably more than 90% and ideally more than 98%.Such oils may be used in the preparation of a medicament or dietarycomposition for the above purposes, in which may be included:

(1). Oils to be added to or used with any type of food for humans oranimals, including but not limited to margarines, modified butters,cheeses and cheese spreads, milks, yogurts, chocolates, chocolate barsand any other types of sweet or candy, snacks, salad oils, cooking oils,cooking fats, bakery products, pates, meats, fish and seafood products,infant milks and infant foods of all sorts, canned or bottled productssuch as whips or creams, solid forms such as powders or granules, drinksof any sort and cereals. (2). Oils to be used in manufacturing any formof nutritional supplement preparation, including but not limited to hardor soft gelatin capsules (whether uncoated or for exampleenteric-coated), granules, tablets, draguees, pastilles, canned orbottled liquids, oils, whips, creams (whether or not in dosage-meteredcontainers) or any other dosage form appropriate to a nutritionalsupplement, including ones for lingual or sub-lingual administration.

(3). Oils to be used in formulae for special nutritional purposesincluding foods and fluids for term and pre-term infants, enteral,parenteral, rectal, percutaneous compositions and foods or fluids forany appropriate route of administration.

(4). Oils to be used in the treatment of any disease in which GLA isbeneficial by oral, lingual and sub-lingual, enteral, parenteral(sub-cutaneous, intra-muscular, intra-venous, intra-peritoneal or otherroute), rectal, vaginal, percutaneous or other routes of administration.The oil may be contained in any of the dosage forms specified in (2) orin any other appropriate dosage form.

(5). Oils to be used in any preparation applied to the skin for thepurpose of caring for normal skin or for treating rough, dry or ageingskin, or for treating injured or burned skin, or skin affected by anydisease or trauma.

(6). Oils to be used in any preparation applied to the hair for thepurpose of caring for normal hair, or for stimulating hair growth, orfor treating thinning, damaged, dry or greasy hair, or for treating hairor the scalp affected by any disease. Preferred oils according to theinvention comprise at least 20% by weight of DLMG.

The oils may be prepared by any method. The following are some examplesbut do not exclude the use of other methods.

i. Any natural oil containing DLMG, especially EPO, but also borage,blackcurrant or other plant oils, and microbial oils, may be treated toseparate out the DLMG. The separated DLMG may be used to enhance orsupplement any oil, natural or synthetic, to make a product containing20%, desirably 25% or more DLMG. Such separation techniques include, butare not limited to low temperature precipitation of less polyunsaturatedtriglycerides (TGs), enzymic digestion using specific enzymes for TGsother than DLMG, differential solution using solvents of more or lesspolyunsaturated TGs, or chromatography techniques using appropriatecolumn conditions, packing, temperatures and pressures and solvents.

ii. Any natural oil whether known or unknown to date, or developed fromknown plants such as Oenothera or other appropriate species by plantbreeding, genetic engineering or other techniques, which contains 20%,desirably 25% or more DLMG.

iii. Any oil made synthetically from GLA and LA. GLA and LA may besynthesised or prepared from any appropriate source. As examples, theymay be made by hydrolysis of natural oils, followed by concentration ofthe GLA and/or LA by techniques known to those skilled in the art suchas urea complexation, low temperature crystallisation, binding tomaterials such as zeolites which selectively concentratepoly-unsaturated fatty acids, differential solution in appropriatesolvents selective for unsaturated or other fatty acids, orconcentrating using enzymes which are selective for particular fattyacids or groups of fatty acids. Appropriate mixtures of GLA or LA or anyappropriate derivatives such as salts, alcohols, amides or othercompounds, may then be converted to tri-glycerides by a range ofdifferent techniques known to those skilled in the art. Examples of suchtechniques include conventional organic synthesis, using for examplezinc as a catalyst, or the use of the chlorides as starting materials,in an appropriate organic solvent with pyridine, or by using anappropriate enzyme system such as lipozyme IM20 (available from Novo) orby using chemical techniques developed for specific placement of GLA orany other fatty acid in a specific position in the tri-glyceridemolecule. The DLMG or LGG resulting from such synthetic procedures canthen be concentrated and purified as appropriate as outlined under (i)above.

It is to be understood that the concept of using DLMG and/or LGG fornutritional, pharmaceutical, skin care or other purposes is independentof the methods which may actually be used for making DLMG or LGG inappropriate forms.

EXAMPLES

1. An oil to be made available for use by food manufacturers containing30%, 45%, 70%, 95%or 99% or DLMG.

2. An oil to be made available for use by food maufacturers of skin careand hair care preparations containing 30%, 45%, 70%, 95% or 99% of DLMG.

3. Ampoules prepared for addition to enteral foods or to sterile lipidemulsions for intra-venous administration, each containing 10 or 20 mlof an oil containing 95% or 99% DLMG.

4. Enteral foods or infant foods containing 1%, 2%, 5% or 10% of an oilcontaining 30%, 45%, 70%, 95% or 99% of DLMG.

5. Skin care preparations containing 0.5%, 1%, 2%, 5% or 10% or 20% ofan oil which contains 30%, 45%, 70%, 95% or 99% of DLMG.

6. Preparations as in (5) but for hair care or scalp care.

7. Oils for direct ingestion or for mixing with other foods such assalad oils containing 30%, 45%, 70%, 95% or 99% of DLMG.

8. Capsules for use as nutritional supplements containing 100 mg, 200mg, 500 mg or 750 mg of an oil containing 25%, 50%, 75%, 90% or 99%DLMG.

9. Capsules as in (8) but for pharmaceutical use.

10. Whips, creams or other formulations contained in bottles, cans orother appropriate containers containing 2%, 5%, 10%, 20% or 50% of oilscontaining 25%, 50%, 75%, 95% or 99% DLMG.

11. Granules, tablets or powders made with dextran, agar, gum acacia,calcium salts or other appropriate vehicle containing oil containing25%, 50%, 70%, 90% or 99% DLMG.

12. Formulations for lingual or sub-lingual administration containingoil containing 25%, 40%, 60%, 80%, 95% or 99% DLMG.

We claim:
 1. A natural or synthetic glycerol oil comprising at least 20% by weight of di-linoleoyl-mono-gamma-linolenyl-glycerol.
 2. The oil of claim 1 containing at least 25% by weight of di-linoleoyl-mono-gamma-linolenyl-glycerol.
 3. The oil of claim 1 containing at least 40% by weight of di-linoleoyl-mono-gamma-linolenyl-glycerol.
 4. The oil of claim 1 containing at least 60% by weight of di-linoleoyl-mono-gamma-linolenyl-glycerol.
 5. The oil of claim 1 containing at least 90% by weight of di-linoleoyl-mono-gamma-linolenyl-glycerol.
 6. The oil of claim 1 containing at least 98% by weight of di-linoleoyl-mono-gamma-linolenyl-glycerol.
 7. A pharmaceutically or nutritionally acceptable oil according to claim
 1. 8. An oil which is enriched or supplemented by the oil of claim
 1. 9. A pharmaceutical composition comprising the oil of claim 1 together with a pharmaceutical carrier.
 10. Di-linoleoyl-mono-gamma-linolenyl-glycerol. 